MULTITARGET DRUGS: VERIFICATION-OF-PRINCIPLE STUDIES

多靶点药物：原理验证研究

• 批准号: 6033211
• 负责人: ALEXANDER J VARSHAVSKY
• 金额: $ 15.6万
• 依托单位: CALIFORNIA INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-01-06 至 2001-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6033211
• 关键词: Saccharomyces cerevisiae; chemical binding; chemical structure; chemical structure function; cytotoxicity; drug design /synthesis /production; gene expression; methotrexate; novobiocin; trimetrexate

The insufficient selectivity of drugs is a bane of present-day therapies. This problem is significant for antibacterial drugs, difficult for antivirals, and utterly unsolved for anti-cancer drugs, which remain ineffective against major cancers, and in addition cause severe side effects. T he problem may be solved if therapeutic agents could acquire a multi-target, combinatorial selectivity, killing (or otherwise modifying) a cell if, and only if, it contains a predetermined set of molecular targets and lacks another predetermined set of targets. Recently, a solution to the problem of drug selectivity was proposed that is applicable to small (less than or equal to 1 kD) compounds (Varshavsky, A. PNAS 95, 2094-2099 (1998)). Two ideas, interference/co-dominance (IC) and anti-effectors, should allow a therapeutic regimen to possess combinatorial selectivity, in which the number of positively and negatively sensed macromolecular targets can be two, three, or more. The in vivo effects of such drugs would be analogous to the outcomes of the Boolean operations "and", "or" "not", and combinations thereof. The proposed studies, to be supported by the present R21 application, will verify the principles of multi-target designs using the yeast Saccharomyces cerevisiae as a model organism. Specific Aims 1) The ubiquitin fusion technique will be used to express and analyze, in the yeast S. cerevisiae, a set of multi-moiety linear peptides that act as IC- type compounds in this in vivo setting. 2) Bipartite, IC-type compounds that comprise the anhydrotetracycline (at) moiety and either the methotrexate (mtx) or the trimetrexate (tm) moiety linked to at will be synthesized, and thereafter characterized in vitro (mutual exclusivity of binding to the purified TetR or DHFR) and in vivo (differential toxicity to S. cerevisiae that either lack or express the at- binding E. coli TetR repressor). 3) Bipartite, IC-type compounds containing the novobiocin (nov) moiety and either the methotrexate (mtx) or the trimetrexate (tm) moiety will be synthesized and tested for differential cytotoxicity with S. cerevisiae that either lack or express the nov-binding B-subunit of E. coli DNA gyrates (GyrB). 4) Tripartite, IC-type compounds containing the at moiety, the beta- ecdysone (ec) moiety, and either the methotrexate (mtx) or the trimetrexate (tm) moiety will be synthesized and tested for differential toxicity with S. cerevisiae that lack or express the corresponding macromolecular targets and their pairwise combinations.

药物的选择性不足是当今治疗的一个祸害。这个问题对于抗菌药物是重要的，对于抗病毒药物是困难的，并且对于抗癌药物是完全未解决的，其对主要癌症仍然无效，并且另外引起严重的副作用。不 如果治疗剂能够获得多靶点、组合选择性，并且当且仅当细胞含有预定的一组分子靶点并且缺乏另一组预定的靶点时杀死（或以其它方式修饰）细胞，则可以解决该问题。最近，提出了药物选择性问题的解决方案，其适用于小（小于或等于1 kD）化合物（Varshavsky，A. PNAS 95，2094-2099（1998））。干扰/共显性（IC）和抗效应子这两个概念应该允许治疗方案具有组合选择性，其中正和负感测的大分子靶标的数量可以是两个、三个或更多个。这些药物的体内作用类似于布尔运算“与”、“或”不”及其组合的结果。建议的研究，由目前的R21应用程序的支持，将验证多目标设计的原则，使用酵母酿酒酵母作为模式生物。具体目的1）利用泛素融合技术在酵母S.酿酒酵母，一组多部分线性肽，其在该体内环境中充当IC型化合物。2)将合成包含脱水四环素（at）部分和与at连接的甲氨蝶呤（mtx）或曲美蝶呤（tm）部分的双组分IC型化合物，然后在体外（与纯化的TetR或DHFR结合的互斥性）和体内（对S.酿酒酵母缺乏或表达at-结合E. coliTetR阻遏物）。3)将合成含有新生霉素（nov）部分和甲氨蝶呤（mtx）或三甲曲氨蝶呤（tm）部分的双组分IC型化合物，并测试与S.酿酒酵母，其缺乏或表达E. coliDNA螺旋体（GyrB）。4)将合成含有at部分、β-蜕皮激素（ec）部分和甲氨蝶呤（mtx）或三甲曲氨蝶呤（tm）部分的三组分IC型化合物，并测试其与S.酿酒酵母的大分子靶点，以及它们的成对组合。